PROJECT SUMMARY Creatine (Cr) and phosphocreatine (PCr) are two major metabolites of the creatine kinase reaction that play vital roles in muscle energetics. However, existing methods for detecting Cr or PCr e.g. proton and phosphorus magnetic resonance spectroscopy (1H and 31P MRS), suffer from low sensitivity, low spatial resolution, and partial volume effect. Moreover, 1H MRS can measure only total Cr (tCr=Cr+PCr) and cannot differentiate PCr and Cr, and thus has little relevance for measuring energy metabolism. Chemical exchange saturation transfer (CEST) is a novel approach to increase sensitivity to metabolites in solution, which applies frequency selective RF pulses to saturate exchanging protons and detect subsequent changes in water signals. In addition, CEST signals from Cr and PCr arise at different frequency offsets from water (2 ppm and 2.7 ppm, respectively), which provides the ability to image these two metabolites separately. However, although CEST has shown promise and has attracted a lot of attention, there has been almost no validation that CEST data reflect actual metabolite levels, and no comprehensive evaluation of the specificity and sensitivity for mapping Cr and PCr in vivo. First, for CEST imaging of Cr at 2 ppm (CEST@2ppm), although Cr has guanidine amine protons at 2 ppm, protein arginine residues have similar chemical shifts (?2 ppm), and thus may confound measurements of Cr. In previous validation of CEST imaging of Cr, only contributions from the major tissue metabolites were considered, but contributions from proteins were ignored. This may be due to that it is difficult to mimic the arginine residues of proteins using simple model phantoms because there are many types of proteins which contain different proportions of arginine residues in biological tissues. Second, attempts to evaluate CEST imaging of Cr in vivo have been also previously performed in muscle following exercise. However, due to the quick recovery of Cr content in muscle (?2 mins) following exercise, it is hard to acquire high SNR and reliable signals for evaluating its specificity and sensitivity. These two challenges hinder its validation and limit its applications. The proposed study will validate the use of CEST MR imaging Cr and PCr in muscle through addressing these two challenges. In Aim 1, we will use dialysis to remove Cr and other small molecules from samples of muscle tissue homogenates to investigate the influence of proteins on CEST@2ppm in muscle. Together with measurements on phantoms containing major metabolites, the dialysis of tissue homogenates can provide a comprehensive investigation of the origins of CEST signals. In Aim 2, we will use Guanidinoacetate N-Methyltransferase deficiency (GAMT-/-) mice with a controlled Cr supplementation which provide an ideal experimental model to vary Cr and PCr concentrations to study its specificity and sensitivity in vivo. Through these two Aims, we will validate the method, which will be very important for future clinical translation and will ultimately improve non-invasive MRI diagnoses of many muscular disorders.